Liver isozyme of rabbit glycogen synthase. Amino acid sequences surrounding phosphorylation sites recognized by cyclic AMP-dependent protein kinase.

Y H Wang,A W Bell,M A Hermodson,P J Roach

doi:10.1016/s0021-9258(19)75975-4

Abstract

Glycogen synthase, the rate-limiting enzyme in glycogen biosynthesis, has been postulated to exist as isozymes in rabbit liver and muscle (Camici, M., Ahmad, Z., DePaoli-Roach, A. A., and Roach, P. J. (1984) J. Biol. Chem. 259, 2466-2473). Both isozymes share a number of properties including multiple phosphorylation of the enzyme subunit. In the present study, we determined the amino acid sequences surrounding phosphorylation sites in the rabbit liver isozyme recognized by cyclic AMP-dependent protein kinase. Two dominant phosphopeptides (P-1 and P-2) were generated from tryptic digestion. Amino acid sequences of the purified peptides were determined by automated Edman degradation using a gas-phase sequenator. The locations of phosphorylated residues were identified by measuring 32Pi release during Edman degradation cycles. The NH2-terminal sequence of peptide P-1 is S-L-S(P)-V-T-S-L-G-G-L-P-Q-W-E-V-E-E-L-P-V-D-D-L-L-L-P-E-V. This sequence exhibits a strong homology to the site 2 region in the NH2 terminus of the muscle isozyme. The NH2-terminal sequence of peptide P-2 is M-Y-P-R-P-S(P)-S(P)-V-P-P-S-P-L-G-S-Q-A. This sequence shows strong homology to the site 3 region in the COOH terminus of the muscle isozyme. However, some interesting sequence differences were revealed in this region. For example, substitution of serine for alanine at position 6 of peptide P-2 created a new phosphorylation site for cyclic AMP-dependent protein kinase. Phosphorylation of the proline/serine-rich site 3 region correlated with inactivation of the liver isozyme and suggests an important role for this segment of the molecule in the regulation of glycogen synthase. No phosphorylation sites corresponding to sites 1a and 1b of the muscle isozyme were detected. In addition, the results provide definitive chemical proof that glycogen synthase from rabbit liver and muscle are isozymes encoded by distinct messages.

Highlights

From the Department ofBiochemistry, Indiana University School of Medicine, Indianapolis, Indiana 46223 ana' the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907
The phosphorylation sites of the muscle enzyme aredistributed between two CNBr fragments, CB-1 and CB-2,which are located in NH, and locations of phosphorylated residues were identified COOH-terminal regions of the enzyme, respectively [16].The by measuring 32Pirelease during Edman degradation amino acid sequences around several phosphorylation sites cycles
The size differences of CNBr fragments between the liver and muscle enzymespresumably reflect differences in theirprimary StNCtures;and we proposed that glycogen synthases from rabbit skeletal muscle and liver wereisozymes [22]

Summary

Introduction

Substitution of serine for alanineat position 6 of peptide P-2 created a new phosphorylation site for cyclic AMP-dependent protein kinase. The amino acid sequences around phosphorylation sites recognizedbycyclic AMP-dependent protein kinase were determined for the rabbit liver isozyme. The first structural indications for the existence of liver and muscle isozymes of glycogen synthase came from comparison of the size of phosphorylated CNBr fragments of the two enzymes from rabbit [22].

Results

Conclusion